Isoenzymes are catalytically active proteins that can exist in multiple forms in the same species at different life stages, in different organs and in different tissues. Certain biologically active enzymes are composed of more than one polypeptide subunit which can appear in the body as a characteristic imbalanced mixture of various isoenzymes. This imbalance produces a characteristic isoenzyme profile which can be associated with a particular physiological condition.
Lactate dehydrogenase (LDH), sometimes called lactic acid dehydrogenase or L-lactate:NAD oxidoreductase, is such a biologically active enzyme. LDH is a tetrameric protein composed of four 35 kilodalton-sized subunits which appears in the human body in five isoenzyme forms. The LDH subunits are composed of two different types of polypeptide chains, commonly called "M" and "H," which can combine and form the homotetramer and heterotetramer isoenzymes, H.sub.4, H.sub.3 M, H.sub.2 M.sub.2, HM.sub.3 and M.sub.4. This nomenclature arises because the M-subunit is prevalent in skeletal muscle and the H-subunit is prevalent in tile heart.
The M-subunit and H-subunit containing isoenzymes originally were identified by electrophoretic separation and were conventionally referred to as LD-1, LD-2, LD-3, LD-4 and LD-5 based on their relative migration towards the anode with LD-1 being the fastest. Their corresponding subunit compositions are H.sub.4, H.sub.3 M, H.sub.2 M.sub.2, HM.sub.3 and M.sub.4 respectively.
As used herein, the term "LDH" refers to lactate dehydrogenase as an enzyme species composed of all its multiple isoenzymes which contribute to its total measurable enzyme (total LDH) activity and which constitute its characteristic isoenzyme profile. The term "LD" refers to an individual isoenzyme of lactate dehydrogenase as specifically identified by the numeric suffix of 1-5. For example, the homotetramer of lactate dehydrogenase composed of H-subunits is the isoenzyme conventionally called LD-1 and the heterotetramer composed of three H-subunits and one M-subunit is the isoenzyme called LD-2.
The proportional amount of each isoenzyme of LDH varies considerably between organs and tissues but is relatively constant within any given organ or tissue. This distribution gives each organ and tissue a characteristic isoenzyme profile or pattern. For example, heart and erythrocytes have high proportions of LD-1 and LD-2, liver and skeletal muscle contain predominantly LD-5, and lung, kidney and brain contain mixtures in which LD-2, LD-3 and LD-4 may predominate to varying degrees. Presence of these isoenzymes from a diseased or damaged organ or tissue in the blood can be detected by an elevation of the total normal level of serum LDH. Further characterization of the serum isoenzyme composition can aid in identifying the physiological source responsible for isoenzymes in the blood. Thus, determinations of serum LDH and its isoenzyme composition are becoming increasingly more clinically valuable in the diagnosis of a variety of pathological disease states.
The diagnosis of suspected myocardial infarction is probably the most frequent clinical application of LDH isoenzyme determinations. In these cases, an increase in the LD-1 isoenzyme relative to the other isoforms can be specific and confirmatory for myocardial damage since the heart contains the largest proportion of the LD-1 enzyme. Speed in the diagnosis of acute myocardial infarction is desirable and often necessary to save the patient's life.
Electrophoresis was the first method used to separate the LDH isoenzymes, and is still widely used to obtain complete isoenzyme profiles. This method has several disadvantages in that it requires multiple and lengthy steps, and expensive equipment. The procedure typically requires at least about one hour to complete, with much of that time dedicated to sample manipulations by a trained technician. Separations using ion exchange columns have also been developed, but also have similar disadvantages.
An alternative approach to the analysis of LDH isoenzymes has been to selectively inhibit or denature some of the isoenzymes and so quantitate the relative proportion of those isoenzymes resistant to such treatment. For example: urea, heat and high pH have been used to progressively inactivate those isoenzymes containing the most M-subunits; high substrate levels of pyruvate or lactate have been used to selectively inhibit the H-subunit; and alpha-ketobutyric acid has been used as a substrate having a specificity for the H-subunit.
In U.S. Pat. No. 4,250,255, an approach is described where the total LDH activity is first measured, and then the isoenzymes are selectively inhibited by treating the sample with an ionic amphiphile. The treated sample is then measured for enzyme activity and that measurement is subtracted from the first measurement. The difference constitutes the activity of the uninhibited isoenzyme.
However, none of the foregoing methods can quantitate the complete isoenzyme profile of lactate dehydrogenase quickly from a single sample of biological fluid without subjecting the sample to various tedious and time-consuming manipulations, such as dilutions, pretreatments and the like or to further multi-step assays. Under clinical situations requiring relatively rapid diagnosis within minutes for treatment of a pathological state, such as in an emergency room or in a delivery room, speed and minimal sample preparation is essential.
There is a long standing clinical need for a rapid assay method and assay reagent capable of determining within minutes the completed isoenzyme profile in a biological fluid suspected of containing a biologically active enzyme, such as LDH present. This invention provides for such an assay method and assay reagent system which requires substantially no sample manipulation prior to assaying and is over 10-fold faster than present electrophoretic methods.